Acantharnoebae are opportunistic protozoa responsible for encephalitis, corneal infections in contact lens wearers, and disseminated infections in AIDS patients. A fourth of all isolates recovered from clinical and environmental sources harbor host-specific non-culturable endosymbionts, some of which are related to chlamydiaceae and rickettsieae on the basis of ribosomal sequence analysis. Preliminary evidence suggests that certain of these symbionts may augment amoebic virulence. An underlying hypothesis of this proposal is that long term co-evolution maximizes overall fitness of the symbiotic relationship which may, in tam, result in increased pathogenic potential of the host. This proposal will further characterize the endosymbiont assemblage known to be present in Acanthamoeba through comparative sequence analysis of rRNA genes to assess overall diversity. Computer designed, taxon-specific fluorochrome-tagged probes will be synthesized for in situ hybridization to catalogue existing symbiont isolates and for direct screening of environmental habitats to identify niche preferences. The recent description of genus- and subgenus-specific probes for Acanthamoeba will also permit us to design an experimental approach for simultaneous in situ analysis of symbiont and host populations from natural habitats to discern epidemiologic links to human infection. This project will require two six-month sojourns at the Technical University in Munich, the first for completion of the rRNA sequencing, probe design work, and evaluation of existing isolates and the second for the examination of specimens from natural habitats and design of Acanthamoeba probes, punctuated by a one month return to the University of Washington for data analysis and preparation of a manuscript. Manuscripts based upon the epidemiologic studies will be completed after returning to Seattle. The work from this fellowship should better enable my lab to develop highly sensitive and specific probe reagents for use in identifying pathogenic microorganisms which are either new to medical science or which are fastidious and not easily recovered by conventional techniques.